| 小鼠KGF基因毛囊特异性表达载体的构建及mESC脂质体悬浮转染条件的优化 |
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| 引用本文： | 梁伟,肖红,高笑宇,杜晓媛,汪慧,郭旭东,刘东军.小鼠KGF基因毛囊特异性表达载体的构建及mESC脂质体悬浮转染条件的优化[J].生物技术通报,2011(11). |
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| 作者姓名： | 梁伟 肖红 高笑宇 杜晓媛 汪慧 郭旭东 刘东军 |
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| 作者单位： | 内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,呼和浩特,010021 |
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| 基金项目： | 国家转基因新品种培育重大专项(2008ZX08008-002) |
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| 摘 要： | 旨在构建小鼠角质细胞生长因子(KGF)真核表达载体pCDsR-UKA,通过脂质体转染法转染小鼠胚胎干细胞( mESC),并进一步优化其转染条件,最终获得可以正常生长并稳定表达红色荧光蛋白的转KGF基因的ES细胞.利用RTPCR技术扩增小鼠KGF基因cDNA并构建表达载体pCDsR-UKA (6.6 kb),经鉴定正确的重组质粒DNA用脂质体包裹后转染mESC.从小鼠成纤维细胞cDNA扩增出891 bp的KGF基因片段与UHS启动子和BGH polyA序列成功重组到pCDsRed2载体中.经酶切和DNA测序验证,插入载体的DNA片段为KGF基因且方向正确.采用脂质体法优化转染条件,mESC最高转染效率达到(34.4±4.1)％.经G418筛选的转基因ES细胞通过PCR鉴定证实外源基因已整合在ES细胞基因组中.成功获得了小鼠KGF基因片段,以及真核表达载体pCDsR-UKA,经优化的脂质体悬浮法转染条件,在六孔板中当DNA与脂质体比例为3:10时,可获得最佳转染效率且不改变ES细胞的生长状态,经筛选获得了转基因ES细胞克隆.为下一步通过四倍体补偿技术获得ES小鼠提供了转基因ES细胞.
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| 关 键 词： | KGF 表达载体 胚胎干细胞 毛囊 UHS 红色荧光蛋白 脂质体转染 悬浮法 |
Construction of a Hair-follicle-cell-specific Expression Vector for KGF Gene and Optimization of the Transfection Condition with Liposome on Suspension mESCs |
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| Liang Wei,Xiao Hong,Gao Xiaoyu,Du Xiaoyuan,Wang Hui,Guo Xudong,Liu Dongjun.Construction of a Hair-follicle-cell-specific Expression Vector for KGF Gene and Optimization of the Transfection Condition with Liposome on Suspension mESCs[J].Biotechnology Bulletin,2011(11). |
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| Authors: | Liang Wei Xiao Hong Gao Xiaoyu Du Xiaoyuan Wang Hui Guo Xudong Liu Dongjun |
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| Institution: | Liang Wei Xiao Hong Gao Xiaoyu Du Xiaoyuan Wang Hui Guo Xudong Liu Dongjun(The Key Laboratory for Mammal Reproductive Biology and Biotechnology of Ministry of Education of P.R.China,Inner Mongolia University,Hohhot 010021) |
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| Abstract: | The present study is aimed to construct a eukaryotic expression vector pCDsR-UKA of keratinocyte growth factor(KGF),transfect it into mouse embryonic stem cells(mESCs)with liposome transfection method,and further to optimize its transfection condition,finally to obtain transgenic mESCs which stably express red fluorescence protein and grow normally.Mouse KGF gene cDNA fragment was amplified with RT-PCR and the expression vector pCDsR-UKA(6.6 kb)was constructed with molecular cloning technique.Identified rec... |
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| Keywords: | Keratinocyte growth factor Expression vector Embryonic stem cell Hair follicle Ultra-high sulfur keratin Red fluorescent protein Liposome transfection Suspension method |
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